The long term objective of this proposal is to understand the role of the retinoblastoma (RB) gene, namely loss of RB function, in the development of human tumors as well as its potential to be a prognostic indicator of the clinical outcome in patients with tumors in which the loss of RB function has occurred. Initial studies will concentrate on such tumors as cancers of the lung, breast, bladder, bone, ovary and kidney as well as acute myeloblastic leukemia. Whenever possible normal tissue from the same site will be examined as will metastatic lesions from the same patient when available. Major emphasis will be placed on examining the RB status of human tumors at the single cell level in frozen and paraffin sections as well as direct Western immunoblotting, although many of the tumors will be examined in addition at the RNA and DNA levels. The latter will be done in our attempt to find the structural changes that have occurred within the RB gene to cause the loss of function and to confirm and complement our protein studies. The use of paraffin sections will be done whenever possible since we have recently been successful in obtaining excellent immunohistochemical RB staining of paraffin sections using our polyclonal RB antibody, which, to our knowledge, is not possible with any commercially available RB antibody, whether polyclonal or monoclonal. The fulfillment of the goals of this proposal should provide a much better understanding of the role of the RB gene in some of the most common human malignancies and equally or perhaps even more importantly determine whether monitoring the RB status in patients with specific malignancies will provide significant information related to their clinical outcome or the aggressiveness of their disease.